POS0095 CaMK4 CONTROLS T REGULATORY CELL METABOLISM AND DEFINES THEIR FUNCTION AND STABILITY IN SYSTEMIC LUPUS ERYTHEMATOSUS
Authors: M. Scherlinger, W. Pan, R. Hisada, A. Boulougoura, M. Vukelic, G. Tsokos
Journal: Annals of the Rheumatic Diseases (2022)

Background Calcium/Calmodulin-dependent protein kinase IV (CaMK4) is a serine kinase expressed in CD4 T cells whom activity is increased in patients with active systemic lupus erythematosus (SLE). We have shown that CaMK4 negatively impacts T regulatory (Treg) cells differentiation and function in SLE, but the underlying mechanism remains unclear. Recent, data suggest that CaMK4 impacts cellular metabolism. Objectives Investigate how CaMK4 impacts Treg cell metabolism and its potential effect on Treg cell function. Methods We harvested CD62L + CD4 + T cells from wild-type (WT) or Camk4 -/ - mice and differentiated them in vitro into Treg (iTreg) cells. We assessed iTreg metabolism using Seahorse XF analyzer and mass spectrometry (metabolomics). Gene expression was assessed at the mRNA (RT-qPCR) and at the protein level (Western Blot). Phosphofructokinase activity was assessed by a colorimetric assay (Abcam). In vitro gene knockdown was conducted by transfecting a guide RNA (gRNA) in CRISPR/Cas9-expressing T cells. Treg cell function was evaluated by in vitro immunosuppressive assay and in vivo by the adoptive transfer of T conventional T and iTreg cells (8:1 ratio) in Rag1 -/ - mice to induce inflammatory colitis. The relevance of CaMK4 in SLE was evaluated in vivo using a T-cell specific knockdown of CaMK4 in the B6.lpr mouse model, and in humans by culturing SLE patient T cells with KN-93, a CaMK4 specific inhibitor. Results iTreg cells from Camk4 -/ - mice had decreased glycolysis and increased mitochondrial metabolism compared to WT mice. Metabolomics studies suggested decreased activity of the rate-limiting glycolysis enzyme phosphofructokinase platelet-type (PFKP). While PFKP mRNA and protein levels were similar between WT and Camk4 -/ - iTreg, we found that PFKP activity was significantly decreased in Camk4 -/ - iTreg, suggesting post-transcriptional control of PFKP activity. Mechanistically, immunoprecipitation experiments confirmed that CaMK4 interacted with PFKP, and phosphoproteomic study suggested that CaMK4 phosphorylated serine residue 539 of PFKP, a site known to control PFKP activity. Excitingly, PFKP’s endproduct fructose 1,6-biphosphate negatively regulates the activation of the mitochondrial metabolism masterswitch AMPK, therefore linking decreased PFKP activity/glycolysis with increased mitochondrial metabolism in Camk4 -/ - Treg. To confirm the importance of PFKP in Treg biology, we confirmed that PFKP knockdown significantly improved iTreg function in vitro (p &lt; 0.01) and in vivo using an adoptive CD4+ T cell transfer in to Rag1 -/ - mice (colitis model). Interestingly, iTreg lacking PFKP were transferred Rag1 -/ - mice were less likely to lose FoxP3 expression and to produce IL-17A, demonstrating higher Treg stability in an inflammatory environment. On a translational basis, lupus-prone B6. lp r mice with a T-cell specific CaMK4 knockdown displayed significantly less lupus manifestations. In human SLE, CD4 + T cells had higher PFKP activity compared to healthy donors, and PFKP activity correlated with the SLE disease activity index (SLEDAI, r= 0.47; p &lt;0.05). Finally, culture of SLE CD4+ T cells with KN-93 led to a significant decrease in PFKP activity (p &lt; 0.001). Conclusion Increased CaMK4 activity in human SLE mediates Treg dysfunction and instability by altering PFKP activity. Restoring normal Treg metabolism by inhibition of CaMK4 or its downstream target PFKP represents a novel strategy for the treatment of SLE. Acknowledgements Marc Scherlinger is financially supported by the Societe Françaises de Rhumatologie (SFR), Philippe, Monahan &amp; Arthurs Sachs foundations. Disclosure of Interests None declared